Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes. Both aging and the presence of coronary atherosclerosis are associated with impaired effective release of endothelial nitric oxide (NO), a known vasodilator and inhibitor of platelet function. Genetic variations of eNOS have been estimated to contribute to at least 30 percent of the variance in plasma NO levels. Select polymorphic variants have been associated with coronary spasm and myocardial infarction, however, the role of eNOS mutations in platelet-derived NO release or its contribution to hemostasis has not been defined. In preliminary data we demonstrate that platelet-dependent NO production decreases with age and stimulated platelets from normal donors with the eNOS 894-GIT gene polymorphism have attenuated release of platelet-derived NO, enhanced platelet aggregation, as well as impaired vasodilation. The central hypothesis of this proposal is that select genetic variations of the eNOS gene contribute to the regulation of platelet-derived NO release and are associated with alteration in platelet function and modulation of vascular hemostasis. To test this hypothesis, we propose: 1) To further define the contribution of relevant eNOS polymorphisms to vascular reactivity, platelet function and NO release as well as determine the impact of aging and ethnicity; 2) To determine the biochemical mechanism(s) for changes in NO in subjects with relevant genetic variants; and 3) To determine the molecular mechanism(s) by which eNOS genetic variants regulate platelet-derived NO release by transfecting megakaryocytic cells with relevant eNOS mutations. Thus, this proposal will use a combination of molecular, cellular, and human studies to define the mechanism(s) by which clinically relevant variations of the eNOS gene regulate platelet-derived NO release and alter platelet function as well as examine the impact of aging on these changes. This proposal is a continuation of the project "Calcium, aging, and hypertension." During years 8 and 9 of this proposal, the original Principal Investigator, Dr. Darrell Abernethy, extended his initial findings by defining the impact of cardiovascular drugs and aging on peripheral vascular reactivity and described the impact of the 894-Gil eNOS polymorphism. We have continued to examine the imoortance of this mutation to the vascular response.